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. 2004 Nov;78(22):12243-51.
doi: 10.1128/JVI.78.22.12243-12251.2004.

Conditionally replicating adenoviruses kill tumor cells via a basic apoptotic machinery-independent mechanism that resembles necrosis-like programmed cell death

Mohamed A I Abou El Hassan  1 , Ida van der Meulen-Muileman, Saman Abbas, Frank A E Kruyt
Affiliations

Conditionally replicating adenoviruses kill tumor cells via a basic apoptotic machinery-independent mechanism that resembles necrosis-like programmed cell death

Mohamed A I Abou El Hassan et al. J Virol. 2004 Nov.

Abstract

Conditionally replicating adenoviruses (CRAds) represent a promising class of novel anticancer agents that are used for virotherapy. The E1ADelta24 mutation-based viruses, Ad5-Delta24 [CRAd(E3-); E3 region deleted] and infectivity-enhanced Ad5-Delta24RGD [CRAd(E3+)] have been shown to potently eradicate tumor cells. The presence of the E3 region in the latter virus is known to improve cell killing that can be attributed to the presence of the oncolysis-enhancing Ad death protein. The more precise mechanism by which CRAds kill tumor cells is unclear, and the role of the host cell apoptotic machinery in this process has been addressed only in a limited way. Here, we examine the role of several major apoptotic pathways in the CRAd-induced killing of non-small-cell lung cancer H460 cells. As expected, CRAd(E3+) was more potent than CRAd(E3-). No evidence for the involvement of the p53-Bax apoptotic pathway was found. Western blot analyses demonstrated strong suppression of p53 expression and unchanged Bax levels during viral replication, and stable overexpression of human papillomavirus type 16-E6 in H460 cells did not affect killing by both CRAds. CRAd activity was also not hampered by stable overexpression of anti-apoptotic Bcl2 or BclXL, and endogenous Bcl2/BclXL protein levels remained constant during the oncolytic cycle. Some evidence for caspase processing was obtained at late time points after infection; however, the inhibition of caspases by the X-linked inhibitor of apoptosis protein overexpression or cotreatment with zVAD-fmk did not inhibit CRAd-dependent cell death. Analyses of several apoptotic features revealed no evidence for nuclear fragmentation or DNA laddering, although phosphatidylserine externalization was detected. We conclude that despite the known apoptosis-modulating abilities of individual Ad proteins, Ad5-Delta24-based CRAds trigger necrosis-like cell death. In addition, we propose that deregulated apoptosis in cancer cells, a possible drug resistance mechanism, provides no barrier for CRAd efficacy.

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Figures

FIG. 1.
FIG. 1.
Infectivity-enhanced AdΔ24RGD[CRAd(E3+)] is more potent in killing H460 cells than AdΔ24[CRAD(E3−)]. The viability of H460 cells was determined by the WST-1 assay, with three experiments. Data show means ± standard deviation (A) and crystal violet staining (B) at 5 and 11 days postinfection, at different MOIs.
FIG. 2.
FIG. 2.
The p53-Bax apoptotic pathway is not involved in CRAd(E3+)- or CRAd(E3−)-induced cell killing of H460 cells. Western blot analyses of p53 and Bax expression in H460 cells infected with CRAd(E3+) or CRAd(E3−) at an MOI of 25 at different time points after infection are shown. As a control, H460 cells were treated with CDDP or with FasL+CHX (Fas). β-Actin serves as a control for loading (A). The reduced expression level of p53 in stable HPV16-E6-overexpressing H460 cells (H460-E6) was confirmed (B). CRAd-induced cell killing at different MOIs was examined in H460-E6 cells and in empty vector-transfected control H460 cells (H460-neo) at 7 days after infection by WST-1 assays. The results obtained for CRAd(E3+) are shown and were similar to those for CRAd(E3−) (C). Values are means ± standard deviation of three experiments.
FIG. 3.
FIG. 3.
CRAd(E3±)-induced cell death is not mediated by caspases and is not dependent on the activation of the mitochondrial apoptotic pathway regulated by Bcl2. H460 cells were infected at an MOI of 25, and cell extracts were made at different times postinfection and subjected to Western blotting. As a control, H460 cells were treated with agonistic Fas Abs in combination with CHX (Fas). (A) The expression levels of unprocessed procaspase-9, -8, and -3 were assessed together with the cleavage of caspase substrate PARP. (B) Bcl2 and BclXL expression was also determined. (C) CRAd(E3+)-induced cell killing in H460-derived stable transfected cell lines overexpressing antiapoptotic Bcl2 and BclXL or the caspase-inhibitor XIAP when compared to the empty vector controls H460\PEFPGK3 and H460\pcDNA3 was studied. Different MOIs were used, and WST-1 activity was measured at 4 days postinfection. Similar results were obtained for CRAD(E3−). (D) Cotreatment with the broad caspase inhibitor zVAD-fmk failed to protect H460 cells from CRAd(E3±)-induced cell death, whereas it was effective in protecting against apoptosis induced by FasL+CHX cells that served as a positive control. Values are means ± standard deviation of three experiments.
FIG. 4.
FIG. 4.
Lack of apoptotic features in H460 cells undergoing cell death triggered by CRAd(E3+) and CRAd(E3−). (A) H460 cells were infected with both CRAds at an MOI of 25 alone or in combination with 100 μM zVAD-fmk for 2 days. As a control, cells were exposed for 16 h to FasL+CHX with or without zVAD-fmk. Nuclei were stained with Hoechst 33342 to visualize chromatin condensation, nuclear shrinkage, or fragmentation that are markers for apoptosis by immunofluorescence microscopy. CRAd-infected cells did not display nuclear apoptotic features, in contrast to Fas+CHX-exposed cells that show condensed and fragmented nuclei reversible by zVAD-fmk. (B) DNA fragmentation was also analyzed, revealing no apoptotic DNA laddering in CRAd-infected cells, whereas FasL+CHX treatment results in clear DNA smearing. (C) Annexin V staining caused by PS externalization was examined 1 and 2 days postinfection and in FasL+CHX-treated H460 cells with and without zVAD-fmk. Cells were cotreated with PI to allow the distinction between Annexin V staining in the presence or absence of an intact membrane detected by immunofluorescence microscopy; the phase-contrast image is also shown (Phase). A minor portion of CRAd-infected cells were PI positive (indicated by arrows). Annexin V staining is caspase independent in CRAd-infected cells and caspase dependent when induced by FasL+CHX. Results are shown for CRAd(E3+)-infected cells; similar findings were obtained for CRAd(E3−)-infected cells.
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